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Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase


We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12–C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3–C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.

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Figure 1: Maximum turnover rates (mol substrate/min/mol enzyme) for P450 BM-3 wild type and 139-3.
Figure 2: Maximum rates reported for alkane hydroxylation by alkane monooxygenases.
Figure 3: Optical spectra for 139-3 and wild-type P450 BM-3.
Figure 4: Positions of the amino acid substitutions in P450 BM-3 mutant 139-3.

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This work was supported by the National Science Foundation, the Max Kade Foundation (fellowship to A.G.), and by Maxygen. The authors also thank Nathan Dalleska for his assistance with GC/MS, and Ulrich Schwaneberg (International University, Bremen, Germany), Tom Poulos (University of California, Irvine, USA), and Andrew Munro (University of Leicester, UK) for helpful discussions. Correspondence and requests for material should be addressed to F.H.A.

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Correspondence to Frances H. Arnold.

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Glieder, A., Farinas, E. & Arnold, F. Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nat Biotechnol 20, 1135–1139 (2002).

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